Hexamethylmelamine (HMM) is an established antitumor agent effective against human neoplasms such as ovarian adenocarcinoma. Due to limited water solubility, HMM is administered orally, and resulting plasma levels are low and variable. In the rabbit, we have shown low plasma levels following oral administration of HMM (or PMM) to be due to rapid first pass metabolism. Pentamethylmelamine (PMM) was developed as a water soluble HMM analog suitable for intravenous administration. At doses producing significant GI toxicity and neurotoxicity, PMM has failed to show significant antitumor activity or myelosuppression. Both compounds are extensively metabolized, primarily by demethylation. Metabolism is required for antitumor activity, yet the mechanism of action remains undefined and metabolism studies have been limited to reports on demethylation. Neither HMM nor its demethylated metabolites are alkylating agents, and previous attempts to show metabolic activation have been negative. The long-term objectives of this proposal are to fully characterize the metabolism of HMM and PMM in rats and human tumor cell lines, including metabolic activation of HMM (but much less for PMM) we have definitely shown in our preliminary studies. Specific aims are 1) to characterize oxidative metabolism by rat liver microsomes, hepatocytes and human tumor cells including metabolite identification and enzyme involvement, 2) to characterize oxidative metabolic activation of HMM and PMM in these systems and determine the role of amine oxidase (as opposed to cytochrome P-450), 3) to determine if further metabolic activation of intermediates occurs via metabolic conjugation, 4) to study several likely initial oxidative intermediates in the metabolic activation and 5) attempt to relate selective metabolic activation of HMM to antitumor activity by studying cytotoxicity in a human tumor stem cell colony forming assay in the presence and absence of liver metabolizing systems. Methods will include preparation of radiolabeled substrates for use in metabolism and activation (binding) studies, and gc, hplc, and GC/MS analysis of metabolites. These studies will contribute to the use and understanding of these agents by data that HMM is an alkylating agent, and relating metabolism to the antitumor activity of HMM and PMM.